Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in the plasma protein factor VIII. There is no cure for Hemophilia A and treatment consists of replacement therapy using preparations of (purified) plasma or the recombinant protein.
Factor VIII circulates as a non-covalent, metal ion-dependent heterodimer. This procofactor form of the protein contains a heavy chain (HC) comprised of A1(a1)A2(a2)B domains and a light chain (LC) comprised of (a3)A3C1C2 domains, with the lower case a representing short (˜30-40 residue) segments rich in acidic residues (see Fay, “Activation of Factor VIII and Mechanisms of Cofactor Action,” Blood Rev. 18:1-15 (2004)). Factor VIII is activated by proteolytic cleavages at the A1A2, A2B and A3A3 junctions catalyzed by thrombin or factor Xa. The product of this reaction, factor VIIIa, is a heterotrimer comprised of subunits designated A1, A2, and A3C1C2 that functions as a cofactor for the serine protease factor IXa in the membrane-dependent conversion of zymogen factor X to the serine protease, factor Xa (see Fay, “Activation of Factor VIII and Mechanisms of Cofactor Action,” Blood Rev. 18:1-15 (2004)).
Reconstitution studies have shown that the factor VIII heterodimeric structure is supported by both electrostatic and hydrophobic interactions (Fay, “Reconstitution of Human Factor VIII from Isolated Subunits,” Arch Biochem Biophys. 262:525-531 (1988); Ansong et al., “Factor VIII A1 Domain Residues 97-105 Represent a Light Chain-interactive Site,” Biochemistry 45:13140-13149 (2006)), and the inter-chain affinity is further strengthened by factor VIII binding von Willebrand factor (Fay, “Reconstitution of Human Factor VIII from Isolated Subunits,” Arch Biochem Biophys. 262:525-531 (1988); Kaufman et al., “Regulation of Factor VIII Expression and Activity by von Willebrand Factor,” Thromb Haemost. 82:201-208 (1999)). Metal ions also contribute to the inter-chain affinity and activity parameters (Wakabayashi et al., “Metal Ion-independent Association of Factor VIII Subunits and the Roles of Calcium and Copper Ions for Cofactor Activity and Inter-subunit Affinity,” Biochemistry 40:10293-10300 (2001)). Calcium is required to yield the active factor VIII conformation. Mutagenesis studies mapped a calcium-binding site to a segment rich in acidic residues within the A1 domain (residues 110-126) and identified specific residues within this region prominent in the coordination of the ion (Wakabayashi et al., “Residues 110-126 in the A1 Domain of Factor VIII Contain a Ca2+ Binding Site Required for Cofactor Activity,” J Biol Chem. 279:12677-12684 (2004)). A recent intermediate resolution X-ray structure (Shen et al., “The Tertiary Structure and Domain Organization of Coagulation Factor VIII,” Blood 111:1240-1247 (2008)) confirmed this calcium-binding site as well as suggested a second potential site within the A2 domain. This structure also showed occupancy of the two type 1 copper ion sites within the A1 and A3 domains. Earlier functional studies have shown that copper ions facilitate the association of the heavy and light chains to form the heterodimer, increasing the inter-chain affinity by several-fold at physiologic pH (Fay et al., “Human Factor VIIIa Subunit Structure: Reconstruction of Factor VIIIa from the Isolated A1/A3-C1-C2 Dimer and A2 Subunit,” J Biol Chem. 266:8957-8962 (1991); Wakabayashi et al., “pH-dependent Association of Factor VIII Chains: Enhancement of Affinity at Physiological pH by Cu2+,” Biochim Biophys Acta. 1764:1094-1101 (2006); Ansong et al., “Factor VIII A3 Domain Residues 1954-1961 Represent an A1 Domain-Interactive Site,” Biochemistry 44:8850-8857 (2005)).
The instability of factor VIIIa results from weak electrostatic interactions between the A2 subunit and the A1/A3C1C2 dimer (Fay et al., “Human Factor VIIIa Subunit Structure: Reconstruction of Factor VIIIa from the Isolated A1/A3-C1-C2 Dimer and A2 Subunit,” J Biol Chem. 266:8957-8962 (1991); Lollar et al., “pH-dependent Denaturation of Thrombin-activated Porcine Factor VIII,” J Biol Chem. 265:1688-1692 (1990)) and leads to dampening of factor Xase activity (Lollar et al., “Coagulant Properties of Hybrid Human/Porcine Factor VIII Molecules,” J Biol Chem. 267:23652-23657 (1992); Fay et al., “Model for the Factor VIIIa-dependent Decay of the Intrinsic Factor Xase: Role of Subunit Dissociation and Factor IXa-catalyzed Proteolysis,” J Biol Chem. 271:6027-6032 (1996)). Limited information is available regarding the association of the A2 subunit in factor VIIIa, and residues in both the A1 and A3 domains appear to make contributions to the retention of this subunit. Several factor VIII point mutations have been shown to facilitate the dissociation of A2 relative to WT and these residues localize to either the A1-A2 domain interface (Pipe et al., “Mild Hemophilia A Caused by Increased Rate of Factor VIII A2 Subunit Dissociation: Evidence for Nonproteolytic Inactivation of Factor VIIIa in vivo,” Blood 93:176-183 (1999); Pipe et al., “Hemophilia A Mutations Associated with 1-stage/2-stage Activity Discrepancy Disrupt Protein-protein Interactions within the Triplicated A Domains of Thrombin-activated Factor VIIIa,” Blood 97:685-691 (2001)) or the A2-A3 domain interface (Hakeos et al., “Hemophilia A Mutations within the Factor VIII A2-A3 Subunit Interface Destabilize Factor VIIIa and Cause One-stage/Two-stage Activity Discrepancy,” Thromb Haemost. 88:781-787 (2002)). These factor VIII variants demonstrate a characteristic one-stage/two-stage assay discrepancy (Duncan et al., “Familial Discrepancy Between the One-stage and Two-stage Factor VIII Methods in a Subgroup of Patients with Haemophilia A,” Br J Haematol. 87:846-848 (1994); Rudzki et al., “Mutations in a Subgroup of Patients with Mild Haemophilia A and a Familial Discrepancy Between the One-stage and Two-stage Factor VIII:C Methods,” Br J Haematol. 94:400-406 (1996)), with significant reductions in activity values determined by the latter assay as a result of increased rates of A2 subunit dissociation.
Significant interest exists in stabilizing factor VIIIa, since a more stable form of the protein would represent a superior therapeutic for hemophilia A, potentially requiring less material to treat the patient (Fay et al., “Mutating Factor VIII: Lessons from Structure to Function,” Blood Reviews 19:15-27 (2005)). To this end, preparations of factor VIII have been described where mutations have been made in the recombinant protein to prevent the dissociation of the A2 subunit by introducing novel covalent bonds between A2 and other factor VIII domains (Pipe et al., “Characterization of a Genetically Engineered Inactivation-resistant Coagulation Factor VIIIa,” Proc Natl Acad Sci USA 94:11851-11856 (1997); Gale et al., “An Engineered Interdomain Disulfide Bond Stabilizes Human Blood Coagulation Factor VIIIa,” J. Thromb. Haemostasis 1:1966-1971 (2003)). However, it has since been suggested that these types of mutation may not be desirable in a therapeutic factor VIII, because they substantially eliminate means for down-regulation. This situation could yield a prothrombotic condition, which may cause harm. Thus, it would be desirable to enhance the stability of both factor VIII and factor VIIIa, but in a manner that minimizes the likelihood of promoting prothrombotic conditions.
In U.S. Patent Application Publ. No. 20090118184, a number of recombinant factor VIII proteins are identified that possess one or more mutations that result in enhanced stability of both factor VIII and factor VIIIa. These recombinant factor VIII proteins have one or more substitutions of a charged amino acid residue with a hydrophobic amino acid residue at either or both of the A1-A2 or A2-A3 domain interfaces. Despite the improvements made in the stability of recombinant factor VIII proteins (and their active forms, factor VIIIa), the need for further improvements continue to exist.
The intermediate resolution X-ray structures of factor VIII (Shen et al., “The Tertiary Structure and Domain Organization of Coagulation Factor VIII,” Blood 111:1240-1247 (2008); Ngo et al., “Crystal Structure of Human Factor VIII: Implications for the Formation of the Factor IXa:Factor VIIIa Complex,” Structure 16:597-606 (2008)) show close contact between A1 (heavy chain) and C2 (light chain) domains. It was recently reported that a factor VIII variant lacking the C2 domain retained the capacity to bind phospholipid membranes, albeit with a marked reduction in affinity (Wakabayashi et al., “Factor VIII Lacking the C2 Domain Retains Cofactor Activity in vitro,” J. Biol. Chem 285:25176-25184 (2010)), supporting a direct role for the C1 domain in this interaction. Furthermore, deletion of the C2 domain did not grossly alter a number of functional properties including the rate of procofactor activation by thrombin, affinity of factor VIIIa for factor IXa, Km of factor Xase for substrate factor X, or kcat for factor Xa generation. However, this deletion did significantly destabilize the cofactor, as judged by increased rates of activity decay following exposure to elevated temperature or chemical denaturants. While contacts between the A1 and C2 domains of factor VIII appear to contribute to protein, and in particular heterodimer stability, little information is available on specific interactions and their functional significance. It would be desirable, therefore, to identify amino acid residues that can be modified to enhance stability between the A1 (heavy chain) and C2 (light chain) domains.
The present invention is directed to overcoming these and other deficiencies in the art.